RESUMEN
Campylobacter jejuni produces both N- and O-glycosylated proteins. Because protein glycosylation contributes to bacterial virulence, a thorough characterization of the enzymes involved in protein glycosylation is warranted to assess their potential use as therapeutic targets and as glyco-engineering tools. We performed a detailed biochemical analysis of the molecular determinants of the substrate and acyl-donor specificities of Cj1123c (also known as PglD), an acetyltransferase of the HexAT superfamily involved in N-glycosylation of proteins. We show that Cj1123c has acetyl-CoA-dependent N-acetyltransferase activity not only on the UDP-4-amino-4,6-dideoxy-GlcNAc intermediate of the N-glycosylation pathway but also on the UDP-4-amino-4,6-dideoxy-AltNAc intermediate of the O-glycosylation pathway, implying functional redundancy between both pathways. We further demonstrate that, despite its somewhat relaxed substrate specificity for N-acetylation, Cj1123c cannot acetylate aminoglycosides, indicating a preference for sugar-nucleotide substrates. In addition, we show that Cj1123c can O-acetylate UDP-GlcNAc and that Cj1123c is very versatile in terms of acyl-CoA donors as it can use propionyl- and butyryl-CoA instead of acetyl-CoA. Finally, using structural information available for Cj1123c and related enzymes, we identify three residues (H125, G143, and G173) involved in catalysis and (or) acyl-donor specificity, opening up possibilities of tailoring the specificity of Cj1123c for the synthesis of novel sugars.
Asunto(s)
Acetiltransferasas/metabolismo , Campylobacter jejuni/enzimología , Acetilación , Acetiltransferasas/química , Acetiltransferasas/genética , Acetiltransferasas/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía en Gel , Cartilla de ADN , Histidina/química , Modelos Moleculares , Mutagénesis , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
FlaA1 from the human pathogen Helicobacter pylori is an enzyme involved in saccharide biosynthesis that has been shown to be essential for pathogenicity. Here we present five crystal structures of FlaA1 in the presence of substrate, inhibitors, and bound cofactor, with resolutions ranging from 2.8 to 1.9 A. These structures reveal that the enzyme is a novel member of the short-chain dehydrogenase/reductase superfamily. Additional electron microscopy studies show the enzyme to possess a hexameric doughnut-shaped quaternary structure. NMR analyses of "real time" enzyme-substrate reactions indicate that FlaA1 is a UDP-GlcNAc-inverting 4,6-dehydratase, suggesting that the enzyme catalyzes the first step in the biosynthetic pathway of a pseudaminic acid derivative, which is implicated in protein glycosylation. Guided by evidence from site-directed mutagenesis and computational simulations, a three-step reaction mechanism is proposed that involves Lys-133 functioning as both a catalytic acid and base.
Asunto(s)
Proteínas Bacterianas/química , Helicobacter pylori/enzimología , Hidroliasas/química , Oxidorreductasas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Glicosilación , Hidroliasas/genética , Hidroliasas/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Regiones Promotoras Genéticas , Relación Estructura-Actividad , Uridina Difosfato Glucosa Deshidrogenasa/química , Uridina Difosfato Glucosa Deshidrogenasa/genética , Uridina Difosfato Glucosa Deshidrogenasa/metabolismoRESUMEN
WbpP is the only genuine UDP-GlcNAc (UDP-N-acetylglucosamine) C4 epimerase for which both biochemical and structural data are available. This represents a golden opportunity to elucidate the molecular basis for its specificity for N-acetylated substrates. Based on the comparison of the substrate binding site of WbpP with that of other C4 epimerases that convert preferentially non-acetylated substrates, or that are able to convert both acetylated and non-acetylated substrates equally well, specific residues of WbpP were mutated, and the substrate specificity of the mutants was determined by direct biochemical assays and kinetic analyses. Most of the mutations tested were anticipated to trigger a significant switch in substrate specificity, mostly towards a preference for non-acetylated substrates. However, only one of the mutations (A209H) had the expected effect, and most others resulted in enhanced specificity of WbpP for N-acetylated substrates (Q201E, G102K, Q201E/G102K, A209N and S143A). One mutation (S144K) totally abolished enzyme activity. These data indicate that, although all residues targeted in the present study turned out to be important for catalysis, determinants of substrate specificity are not confined to the substrate-binding pocket and that longer range interactions are essential in allowing proper positioning of various ligands in the binding pocket. Hence prediction or engineering of substrate specificity solely based on sequence analysis, or even on modelling of the binding pocket, might lead to incorrect functional assignments.
